SUMMARY The discovery of RNA interference and related small RNA pathways has revolutionized molecular biology and bears potential for new generations of therapeutics. Endogenous small RNAs such as microRNAs (miRNAs) collectively regulate the majority of the human transcriptome, and control important aspects of spatio-temporal development. Dysregulation of the miRNA pathway therefore directly contributes to developmental defects and tumorigenesis. Another group of small RNAs call Piwi-interacting RNAs (piRNAs) are often active during germline development, and preserve genome integrity by silencing selfish elements such as transposons. Despite of the progress made on understanding small RNA pathways, we still lack a clear picture of how each step of the pathway is carried out. Biochemical and structural characterization of the molecular machineries that promote these steps will represent major advances for the field. I have a long-standing interest in small RNA pathways. During my graduate studies at Princeton University with Dr. Laura Landweber, which were funded by a pre-doctoral fellowship by the Department of Defense Breast Cancer Research Program, I uncovered an unknown function for piRNAs to protect DNA against loss during genome rearrangement in Oxytricha. This was one of the first studies to show ?self? recognition by piRNAs, and overturned the dogma that piRNAs only target RNA for repression or DNA for deletion. Then, as a post-doctoral fellow in the laboratory of Dr. David Bartel at the Whitehead Institute, I studied the miRNA pathway, which is the small RNA pathway that dominates in most human cells. Specifically, I identified determinants of primary miRNAs (pri-miRNAs, the precursors of miRNAs) that allow for their efficient processing by the Microprocessor complex, and demonstrated rational, de novo design of artificial miRNA genes. This work was funded by the Damon Runyon Cancer Research fellowship. My long-term goal is to understand how RNA-protein interactions work, with a special focus on small RNA pathways. I would like to pursue this exciting topic as the leader of a research group in an academic institution. To achieve this goal, the overall objectives of this application are to obtain training in protein biochemistry and single-particle cryo-electron microscopy (cryo-EM). This valuable training and experience will lay a solid foundation for me to launch my independent research on RNA-protein complexes. The work proposed here comprises two aims. In Aim 1, which will be completed during the mentored phase, I will leverage my knowledge of pri-miRNAs, the Microprocessor substrate, and harness recent development of cryo-EM, to solve the structure of Microprocessor bound with pri-miRNA, which will provide critical insights into how this complex recognizes its substrate and accurately positions the cleavage. I have already demonstrated that a novel, RNA-based purification strategy allowed the assembly of homogeneous Microprocessor in complex with the substrate, and negative-stain EM shows promising results. My next step is to take advantage of the state-of-the-art cryo-EM technique, which is best suited to solve structures of complexes with a relatively large size. More importantly, this aim will allow me to develop biochemical and structural skills for studying RNA-protein interactions that can be applied to many other RNA-protein complexes, such as the RISC-loading complex proposed in Aim 2. Dr. David Bartel's lab is the ideal place for this training because of its deep expertise in miRNA research, as well as the resources brought in by its affiliation, such as EM facilities at Whitehead Institute, Koch Institute, and the HHMI Research Campus. In Aim 2, which will be the focus of the independent phase, I will fully capitalize the training received in my previous work and that proposed in Aim 1, to solve a long-standing problem at the heart of the small RNA pathways: how does RISC-loading complex work to process pre-miRNA, and help form the mature, silencing-competent RISC complex. Through my previous work, I have obtained extensive molecular biology training. My background and expertise in small RNA pathways ideally positions me for the proposed work. The training and research described in this proposal will allow me to develop a tool-kit of approaches that will form the basis of my independent research, and will provide the platform from which to launch my career as an independent investigator.